Nanoencapsulation of n-butanol extract of Symphytum kurdicum and Symphytum asperrimum: Focus on phytochemical analysis, anti-oxidant and antibacterial activity

Objective(s): The current study’s objectives were to obtain different extracts and essential oils of Symphytum kurdicum and Symphytum asperrimum and to determine the chemical composition, as well as to evaluate free radical scavenging activity (IC50) and minimum bactericidal concentration (MBC), and the effect of liposomal formulation on antimicrobial properties. Materials and Methods: Air-dried powdered aerial parts of S. kurdicum and S. asperrimum were used. The antioxidant and antibacterial properties, essential oil compositions, total phenol, and flavonoid contents of different fractions were determined by DPPH test, disk diffusion assay, gas chromatography-mass spectrometry, Folin-ciocalteu reagent, and colorimetric assay method, respectively. The film hydration method was used to fabricate nanoparticles. Results: GC-MS analysis indicated that hexafarnesyl acetone was a major essential oil component. n-butanol and ethyl acetate extracts of S. kurdicum had the highest anti-oxidant activity. Extracts of both plants showed antimicrobial activity. The extracts’ maximum inhibition zones against Staphylococcus epidermidis were established. A particle size analyzer detected the formulation size of 140 nm. The optimum formulation of liposomes contains the ratio of 75 mg lecithin, 25 mg cholesterol, and 50 mg herbal extract. Despite the nanoparticles’ appropriate particle size, the liposomal extract’s antimicrobial effect was lower than that of the free form. Conclusion: Our findings demonstrated that extracts have significant antibacterial and anti-oxidant activities, attributed to their bioactive constituents.


Introduction
In recent years, pharmacophores acquired from herbals have had an important role in medication discovery. Additionally, they were used for developing antibioticresistant microbial strains in search of novel antimicrobial agents (1). Frequently, there is a correlation between the polyphenolic content of herbs and the antimicrobial or antioxidant effects of plant extract preparations, which could help in the identification of the active ingredients (2). This genus is a member of the Boraginaceae family (3). These herbaceous plants are characterized mainly by large, hairy leaves, and tuberous roots (4).
The essential oil yield of Boraginaceae members is low (5,6). According to the most recent research, plants in the Symphytum genus have numerous pharmacological effects, including antimicrobial, anti-oxidant, and antiinflammatory properties. For example, in a study by Rocha et al., Symphytum officinale L. had a significant antifungal effect against Sclerotinia sclerotiorum (7,8). An in vitro study revealed that S. officinale L. extracts have antimicrobial activity against various bacterial strains, particularly Staphylococcus aureus (9). It could be attributed to the aqueous extract's phenolic content (allantoin, rosmarinic acid, caffeic, and chlorogenic acids) (10,11).
Researchers investigated various carriers for herbal extracts loading, such as manganese nanoparticles and micelles (12,13). Liposomes are some of the most interesting; additional studies have confirmed the effect of liposomal structures on herbal extract efficacy. The spherical form of liposomes composed of phospholipids provides the situation for loading both hydrophilic and lipophilic components that, in addition to biocompatibility and non-immunogenic properties, makes it a good choice for loading herbal extracts. Soybean lecithin is a natural phospholipid for preparation of liposomes with acceptable safety. As studied by Singh et al. liposomal neem (Azadirachta indica) gel composed of soybean lecithin and cholesterol demonstrated in vitro drug diffusion and skin retention equal to 62.178% ± 0.91 and 20.03% ± 0.63, respectively (14). Researchers studied the encapsulation of Laurus nobilis leaf extract in liposomes. The results showed that liposomal encapsulation improved the antimicrobial and anti-oxidant properties of the extract compared with non-encapsulated. They also confirmed the application of nano-formulation as a natural preservative for meat products (15).
The genus Symphytum produces allantoin, phenolic acids, and flavonoids (5) whose antibacterial and antioxidant properties have been recognized (16). The main distribution centers of the genus Symphytum, with 113 species, are humid meadow and lake edge parts of Asia, Europe, and America (3). Most phytochemical and biological studies have been conducted on these species. Different Symphytum species have various therapeutic applications (16). In the present study, we focused on native Iranian medicinal plants, Symphytum species, S. asperrimum and S. kurdicum, the biological properties and phytochemicals of which are unknown. To the best of our knowledge, no research has been published on the two mentioned plants yet. The current study aimed to evaluate different extracts and essential oils in various aspects. The extracts were assessed for phytochemical characteristics, as well as potential antimicrobial and anti-oxidant activities. The other goal of the study was to fabricate the preparation of liposomal formulation of extracts and assess the effect of encapsulation on the antibacterial properties of the extract.

Plant materials, solvent extractions, and fractionations
Aerial parts of S. kurdicum and S. asperrimum were collected from ghasemlou valleys, West Azerbaijan province, and Arasbaran forest, East Azerbaijan province, (Iran May 2019), respectively. The specimens were identified and deposited in the Herbarium of the Faculty of Pharmacy, Tabriz Medical University, Iran (TBZFPH (No. 4072)) and (TBZFPH (No. 4073)). Aerial parts of the collected plants were separately air-dried, powdered, and subjected to soxhlet extraction. n-Hexane and chloroform solvents were used in the extraction process using soxhlet apparatus individually, and then the extraction process continued with the maceration technique. The subsequent solvent was ethanol: water (70:30 V:V). The obtained hydroalcoholic extracts were dried, dispersed in water, then fractionated, initially decanted with ethyl acetate and later with n-butanol. The yielded extracts were dried at low temperature under vacuum via a rotary evaporator (Heidolph, Germany). Dried extracts were stored at -8 °C until analysis.

Extraction of essential oil
Hydrodistillation was conducted separately for both plants using a Clevenger apparatus to extract the essential oil. One hundred grams of air-dried plant materials were weighed into a round 1000 ml flask. Then, 750 ml of distilled water and some glycerol were added to the flask. Extraction was performed for two hours. Ultimately, the essential oils were collected and placed into vials and stored at 4 °C for later analysis.

Gas chromatography-Mass (GC/MS) analysis
The components of the essential oils were assessed using a Shimadzu GCMS-QP5050A equipped with a DB-1 column (60 m × 0.25 mm; film thickness 0.25 ϻm). The split ratio was 1: 24. The primary oven temperature was 50 °C which was kept for 3 min then raised to 270 °C at a rate of 4 °C/min. As a final point, the predicted component's RI values (retention index) were calculated and compared with the valid RI values from the NIST 2018 library. Additionally, the qualified percentage of each compound was reported according to the area under the curve (17).

Antimicrobial activity
A disk diffusion assay was used to examine the antimicrobial activities of S. kurdicum and S. asperrimum. Two gram-positive and two Gram-negative bacteria were used as the test organisms: Staphylococcus aureus Persian Type Culture Collection (PTCC 1337), Staphylococcus epidermidis (PTCC 1435), Escherichia coli (PTCC 1330), Salmonella typhi (PTCC 1074), and a fungal strain, Candida albicans (PTCC 5027). Different extracts were suspended in DMSO solvent; bacterial suspensions were cultivated on the Mueller-Hinton agar medium (0.5 McFarland standard (108 CFU/ml)). 30 ϻl of the extracts were transferred to the blank disks. The standard antibiotic discs and negative control were tetracycline (30 µg per disc), amikacin (30 µg per disc), and DMSO (30 µl per disc), respectively. The average diameter of inhibition zones was recorded after 24 hr of incubation at 37 °C. Furthermore, the extracts' minimum bactericidal concentrations (MBCs) were measured. The microbial concentration in wells was 5*105 CFU/ml, and the concentration of extracts varied from 0.39 mg/ml to 25 mg/ml. After incubation of the 96-well plates at 37 °C for 24 hr, culturing on the Mueller-Hinton agar plates was done to assay the minimum concentrations that could inhibit fungal or bacterial growth (18).

Anti-oxidant activity
Among all extracts, the n-butanol and ethyl acetate extracts were assessed for anti-oxidant ability using the DPPH method with some modification. Briefly, the extracts were dissolved in methanol (1 mg/ml) and serially diluted. Then, the DPPH solution was added to all samples in the 1:1 ratio. Consequently, the samples were shaken and incubated at room temperature for 30 min in the dark, and then the mixture was placed in a 10 mm micro cuvette. The samples' absorbance was determined at 517 nm using a Spectronic Genesys 5 spectrophotometer. Inhibition percentages and average inhibition percentages were calculated to discover the correlation between the extract concentrations and average inhibition percentages. Subsequently, the concentrations which may reduce free radicals up to 50% (RC 50 ) were measured according to the line balances (19).

Total phenol contents
Total phenol content was acquired using the Folinciocalteu reagent. In the first instance, standard solutions of methanol and ethyl acetate extracts were prepared (0.1 mg/ ml), then extract solutions (1 ml), Folin-ciocalteu reagent (200 ϻl), and sodium bicarbonate 2% solution (1 ml) were mixed in the test tubes. Test tubes were centrifuged for 5 min at 16128 G-force and incubated in the darkness for 30 min at 25 °C. The spectrophotometric absorbance of samples was measured at 750 nm. Moreover, gallic acid solutions in the 1-10 mg/ml concentration range were treated as samples to get the standard line equivalence (20).

Total flavonoid contents
Total flavonoid content was acquired using an aluminum chloride colorimetric assay reagent. Ten milligram of n-butanol and ethyl acetate extracts were dissolved in 10 ml of methanol (80%), then 2 ml of samples were added to the mixture of distilled water (400 µl) and the reagent (1 ml), which is formed of sodium acetate (49 M) and aluminum chloride (10 M). The absorbance values of arranged mixtures were determined after 20 min at 410 nm, using the spectrophotometer apparatus. Quercetin was the standard compound in the process. 5, 10, 15, 20, and 25 µg/ ml concentrations of quercetin were prepared and assessed in the same way. As a final point, the total flavonoid content of the extracts was reported as a weight ratio percentage (quercetin equivalent: extract) (20).

Preparation of liposomal formulation
The fabrication method of the liposomal formulation was the film hydration method, according to Plangsombat et al., with some modification (21). Therefore, different ratios of soybean lecithin, cholesterol, and sample extracts of S. asperrimum and S. kurdicum, were dissolved in 8 ml of chloroform to form a clear solution ( Table 5).

Evaporation of chloroform was performed under vacuum by rotary evaporator (Rotavapor R-215, Buchi-Switzerland).
Removing the organic solvent would lead to a homogeny film at the bottom of the round bottom flask. For removing the different molecules of chloroform, N 2 gas was applied. The lipid film was hydrated by water at the temperature of 70 °C, and for better hydration with fine particles, bath sonicator (Bandelin, Berlin, Germany) followed by probe sonicator (Bandelin, Germany) were utilized at 70 °C. The un-entrapped extract was precipitated by centrifugation at 4032 G-force (Eppendorf, Centrifuge 5810R, Hamburg, Germany), and for size reduction, the formulation was filtered through preheated 0.45 and then 0.22 filters (22). The formulation was freeze-dried to be dissolved in the proper amount of water with desired concentration for the antimicrobial test. A particle size analyzer evaluated the liposome's size (Wing SOLD 2101, Shimadzu, Japan). The formulation was diluted using distilled water by the ratio of 10:1 to be detected by the instrument.

Total phenol and flavonoid contents
Total phenolic and flavonoids contents of n-butanol and ethyl acetate extracts of S. asperrimum and S. kurdicum collected from North-West of Iran were presented in Table 3. The results showed variations in total phenolic and flavonoid contents, even in the same parts of the two genera of Symphytum species. n-butanol extract of S. asperrimum had the highest total phenolic (57.3 mg GAE/g extract) and flavonoids contents (31.78 mg QE/g extract).
While n-butanol extract of S. kurdicum contained less total phenolic (42.59 mg GAE/g extract) and flavonoids contents (20.55 mg QE/g extract) than S. asperrimum.

Anti-oxidant activity
In order to determine the anti-oxidant properties of the extracts of the two plants, the DPPH (2, 2-diphenyl-1-picrylhydrazyl) method, a widely used method for determining the scavenging activities of various natural components, was used. It was utilized to measure the in vitro anti-oxidant properties of the extracts of S. asperrimum and S. kurdicum; the higher the free radical scavenging activity, the lower the IC 50 values. For the free radical scavenging activity, IC 50 is defined as the amount of extract required to reduce the primary radical DPPH˙ concentration by 50%. The results of the anti-oxidant properties are shown in Table 4. Increased concentration of the extracts shows a gradual increase in inactivity. The free radical scavenging activity of n-butanol and ethyl acetate extracts of S. kurdicum were 48.053±0.04 µg/ ml and 48.51±0.09 µg/ml, respectively. The free radical scavenging activity of n-butanol and ethyl acetate extracts of S. asperrimum were 53.735±0.03 µg/ml and 51.223±0.02 µg/ml, respectively.

Evaluation of the particle size of liposomes
The preparation of the liposomal formulation was according to the film hydration method. The formulation ingredients were n-butanol extracts of S. asperrimum, S. kurdicum, lecithin, and cholesterol by the ratio mentioned in Table 5. The liposomes were filtered for size reduction, but only formulation four was filtered because it was smaller than others. The sizes shown in Table 5 are representative of particle sizes before filtration. The particle size of liposomes of formulation 1 was in the micrometer range that could   Table 4. Anti-oxidant activity of Symphytum kurdicum and Symphytum asperrimum extracts by DPPH assay Table 5. Ratio of lecithin, cholesterol, and an herbal extract for preparation of liposomal formulation be related to the high content of lecithin. Formulation 2 did not form a homogeny film. The non-homogeny parts developed because of the high content of herbal extract could be observed visually. This might be due to the lower lipid content capacity of particles for forming a homogeny film. Therefore, the optimum formulation of liposomes was formulation four by the ratio of 75 mg lecithin, 25 mg cholesterol, and 50 mg herbal extract. The formulation size was 140 nm, which was detected by a particle size analyzer.

Antimicrobial activity
The two Gram-positive (S. aureus and S. epidermidis), two Gram-negative standard bacterial strains (E. coli and S. typhi), and one fungus (C. albicans) have been used. According to the two-way ANOVA test conducted using GraphPad Prism software diameter of the inhibition zone (DIZ) results, ethyl acetate extract of S. asperrimum showed significant antibacterial activity against S. aureus, E. coli, S. typhi, and C. albicans. Chloroform, ethyl acetate, n-butanol, and nanoencapsulated n-butanol extracts of this plant demonstrated significant antibacterial activity against S. epidermidis (P-value<0.001). n-Butanol extract of S. asperrimum was effective against S. typhi and C. albicans. The antimicrobial activity of the different extracts of S. kurdicum against E. coli and S. aureus was not observed, but there are antimicrobial activities against S. epidermidis, S. typhi, and C. albicans (Figure 1). The effects of nanoencapsulated n-butanol extract of S. asperrimum and S. kurdicum were equal to the results of free n-butanol extracts and did not improve antimicrobial properties (Table 6).
Additionally, more precise data on the antibacterial activities were obtained by determining bactericidal concentration. The minimum bactericidal concentration (MBC; (mg/ml)) of extracts against the three bacteria is shown in Table 6. The ethyl acetate extract of S. asperrimum had the most bactericidal properties against S. epidermidis. MBC accredited the extracts antimicrobial activities data.

Discussion
Several factors, including the type of extraction technique, duration of extraction, temperature, location, soil composition, moisture, and altitude, may influence essential oil components. The difference in the essential oil content between two species of the same genus is predictable, but they may also share many characteristics. The essential oil content of S. kurdicum and S. asperrimum was comparable; however, there is little information on other Symphytum species' essential oil contents from previous studies. A study on the essential oil content of Glendora rosmarinifolia yielded similar results (a member of the Boraginaceae family). Eicosane, phytol, nonanal, β-ionene, α-pinene, and β-caryophyllene were identified as the main components of the essential oil (24). Eicosane and nonanal were identified in the essential oil compositions of Cordia verbenaceae (25). Dibutyl phthalate has been reported as the main essential oil constituent of Anchusa italica from Iran (26). One of the main components, hentriacontane, one of the main identified compounds in the current study, of the essential oils of the herbs, is a hydrocarbon with antiinflammatory effects, reducing inflammatory mediators (TNF-α, IL-6, PGE2, COX-2, and iNOS), as well as activation of NF-κB and caspase-1 in LPS-stimulated peritoneal macrophages (27). This compound also has an antitumor and cytotoxic effect on lymphoma cells (28). This compound is present in large quantities in both plants so that the essential oils of S. kurdicum and S. asperrimum could be used as antiinflammatory agents.
Symphytum genus belonging to the Boraginaceae family is a well-known herbal medicine with pharmacological potential due to its bioactive components (29). Flavonoids and phenolic derivatives are the significant chemical compounds in the Symphytum genus (30). Total phenolic and flavonoid contents in S. asperrimum aerial parts extract were significantly higher than those in S. kurdicum aerial parts extract. Phytochemical components in the different species are different and could affect their pharmacological activities. Secondary metabolites, including flavonoids and phenolic derivatives, are affected by various factors, such as soil composition, irrigation, and climatic conditions (31,32). To the best of our knowledge, phenolics and flavonoids have significant beneficial biological activities such as antitumor (33), anti-inflammatory (34), and anti-oxidative activity (35). Phenolics and flavonoids are free radical scavengers due to this facility (36), reducing agents, singlet oxygen, and hydrogen donors (37). Phenolic components inhibit lipid peroxidation and prevent enzyme oxidation (38). In a study, the total phenolic and flavonoid content of the methanol extract of S. anatolicum was measured, and the results were 32.7 mg GAE/g and 13.3 mg RE/g (39). The total phenolic and flavonoid contents of aerial parts of S. asperrimum and S. kurdicum were higher than those of other species in this genus, according to the bibliographic data.
Due to the many side effects and complications initiated by synthetic constituents, there is a demand for natural extracts or essential oils with anti-oxidant activities, particularly those derived from herbs (40). Recent studies show a positive correlation between anti-oxidant activity and total phenol and flavonoid levels (41). S. asperrimum and S. kurdicum extracts both demonstrated high levels of freeradical-scavenging activity, respectively. The total flavonoid and phenolic contents prove that phenolic components play a significant role in anti-oxidant activity. It is also notable that except phenolics, allantoin, rosmarinic acid, caffeic acid, and their derivatives, as identified in the Symphytum family, might be attributed to the high anti-oxidant activity.
There have been no previous reports on the anti-oxidant activities of S. kurdicum and S. asperrimum. At the same time, previous research on other Symphytum species, such as S. officinale, revealed that its ethanolic extract had high radical scavenging activity against DPPH radicals (39.97 µg/ ml) in comparison with an aqueous extract (96.21 µg/ml) (35). The free radical scavenging activities of methanol and ethyl acetate extracts of S. anatolicum were 2.70 ± 0.15 mg/ ml and 10.57 ± 0.01 mg/ml (42). According to the reports other Symphytum species that have also shown significant anti-oxidant properties (43,44). The potent anti-oxidant properties could be attributed to their phenol and flavonoid contents, confirming that phenolic compounds like caffeic acid derivatives play a prominent role in anti-oxidant activity (39).
The in vitro antibacterial properties of these two Iranian native plants have been investigated for the first time.
According to the results of the DIZ and MBC values, S. epidermidis was the most sensitive microorganism. One of the most influential herbal families with antimicrobial activities is the Boraginaceae (45). Previous research tested the inhibitory effects of methanolic extract of the S. officinale L. leaves against S. aureus, Pseudomonas aeruginosa, Salmonella typhimurium; Shigella sonnei, Klebsiella pneumonia, and Escherichia coli. The antibacterial test results revealed that S. officinale L. extract had antibacterial activity against the tested strains. DIZ was greater than 7 mm (46). The strong antimicrobial activities of the extracts related to the phytochemicals of the Symphytum genus, including phenolic compounds (caffeic acid, allantoin, and luteolin glycoside), are possibly related to the synergetic effects of compounds, as well as to other bioactive components existing in the whole parts of the plant (3). Mangonia et al. investigated the in vitro and in vivo characteristics of phycocyanin liposomal formulation. The liposome structure was composed of soy phosphatidylcholine and cholesterol, and results showed that the preparation method could affect the liposome characteristics. According to their findings, the liposomal formulation was an excellent choice for topical formulation containing phycocyanin as an anti-inflammatory agent, and encapsulation could increase the anti-inflammatory effect dose-dependently. Therefore, a lower concentration of liposomal formulation had an impact equal to the higher concentration of free phycocyanin (47). Aisha et al. evaluated the liposomal formulation of Orthosiphon stamineus extracts containing soybean phospholipids.
Liposome structure could increase the ethanolic extract solubility about four times, and the liposome entrapment efficiency reached 66%. Evaluation of the intestinal absorption showed significant enhancement that could result from higher aqueous solubility of the liposomal extract. The mentioned formulation could be a choice for oral or topical drug delivery (48). Matouskova et al. examined the antimicrobial activities of several herbal extracts encapsulated in different particles and compared the results with non-encapsulated extracts. The prepared particles were stable, and the antimicrobial effect was evaluated against four bacterial strains. The stability of liposomes was higher than chitosan particles, but the inhibitory effect of chitosan particles was very high (49). In contrast to previous studies, the liposomal extract's antimicrobial effect was lower than the free form in the present study. As the liposome contains a bilayer structure, the membrane will lead to the sustainable release of the loaded component that can be a reason for the lower effect of the liposomal form, but confirmation needs further studies.

Conclusion
In this study, the most abundant constituent of both essential oil compositions with beneficial biological effects was a hydrocarbon compound, hentriacontane. Significant anti-oxidant and antimicrobial activities of n-butanol and ethyl acetate extracts were mainly attributed to the high levels of phenolic and flavonoid constituents of S. asperrimum and S. kurdicum. The extracts were encapsulated in the liposomal formulation using the film hydration method, and the size of final liposomes was in the nanometer range; the result of liposomes antimicrobial activity was in conflict with previous studies, and the probable reason might be the existence of bilayer structure of liposome which retards the release of the extract. Further studies should be conducted to confirm the results of the present study. In vivo assessment of anti-oxidant and antimicrobial properties besides the toxicity studies of the extracts of S. asperrimum and S. kurdicum are consequently suggested.